Project Summary Cellular processes are regulated by a complex interplay among different layers of epigenetic information, including DNA methylation, histone modification, nucleosome position, and expression of noncoding RNA. DNA methylation is the best studied epigenetic modification in mammals. The genome-wide DNA methylation profile (i.e., DNA methylome) is established early in development for regulation and perturbed methylome is critically involved in disease development. There have been a number of genome-wide technologies developed for profiling DNA methylomes. Whole genome bisulfite sequencing (WGBS) is generally considered the gold standard for DNA methylation analyses. The approach generates methylomic profile with single-nucleotide resolution. Conventional WGBS requires a large amount of DNA. Although single-cell bisulfite sequencing was demonstrated recently, these assays were based heavily on manipulation in tubes/well plates and manual procedures, making rapid processing of a large number (>1000) of single cells difficult. In this project, we will develop a high-throughput single- cell bisulfite sequencing technology, referred to as Drop-BS, based on a microfluidic droplet platform. We will test and validate the technology by examining intratumoral heterogeneity in single-cell DNA methylomes of breast tumors from patients and patient-derived xenograft (PDX) mouse model.